The overall objective of our research has been to purify and to characterize the glutaminase isoenzymes from rat kidney in an effort to determine their physiological function, their regulation, and their possible contribution to increased renal ammonia synthesis during metabolic acidosis. We have completed determination of their subcellular localization, developed methods for their purification and established that the phosphate-independent glutaminase is a partial reaction of gamma-glutamyltranspeptidase. We now plan to develop the methodology needed to selectively precipitate phosphate-dependent glutaminase from Triton solubilized mitochondria with antibodies and to apply this methodology to determining the relative rates of synthesis and degradation of this glutaminase in normal and acidotic rats. We also plan to use mitochondria in which the phosphate-dependent glutaminase is inhibited by a glutamine affinity label as a system for determining the properties of the mitochondrial transport system for glutamine. We also plan to compare the kinetics of glutaminase inactivation by chloroketone and DON affinity labels and to further characterize the phosphate dependent dimerization of the enzyme.